DE2264764C3 - Process for the production of yeast mutants with high citric acid production capacity - Google Patents

Description

The invention relates to a process for the production of yeast mutants with a high ability to produce citric acid.

Citric acid is found in the food industry, pharmaceuticals, cosmetics and other industries in large amounts of use. Their industrial production through microbial fermentation was limited so far on the use of mold and raw materials containing carbohydrates. In recent years In publications and patents, the possibility of use was also increasingly found unicellular microorganisms such as yeast or bacteria, as well as the use of hydrocarbons described as a raw material for the production of citric acid. For an industrial production In practice, however, these methods are the standard for conventional methods with Aspergillus and carbohydrates still inferior as a substrate. The main reasons for this are:

1. In almost all of the processes described, the citric acid concentrations are in the fermentation suspension still relatively low and this makes work-up sometimes inefficient.

2. In addition to citric acid, iso-citric acid is sometimes formed in considerable quantities, which are below to separate from the citric acid on a large scale only with great difficulty is.

It has also already been found that the citric acid yield can be increased by adding an aconitase inhibitor or citric acid antimetabolites or their precursors in the fermentation medium can. The use of aconitase inhibitors in fermentation is off on an industrial scale price reasons, however, not advantageous. Also with the addition of the aconitase inhibitor there is the fermentation medium the risk that the substance can get into the end product and there for the Is toxic to humans.

It was therefore the aim of the invention that by the Antimetabolite-induced aconitase inhibition by genetic mutation so that the addition an aconitase inhibitor is unnecessary for the fermentation medium.

The invention relates to a process for the production of yeast with high citric acid production capacity, in which citric acid-producing yeasts are treated with mutation-inducing agents, the treated yeasts are inoculated into a glucose-mineral medium, then incubated for several days at 28 ° C. and removed from the Nutrient medium, the enriched yeast mutants with high citric acid production capacity are isolated after plating on complete medium plates according to the usual method, characterized in that in the course of the multi-day incubation at 28 ° C increasing concentrations of citric acid antimetabolites or their precursors up to a final concentration of 5 χ 10 " ² Mol / l added.

Surprisingly, it was found that yeast strains with lower citric acid production capacity even at a lower concentration Aconitase inhibitor, e.g. B. monofluoroacetate, im Growth will be inhibited than those with higher citric acid production. The inventive addition

Hi rate of a citric acid antimetabolite, such as. B. Fluoroacetic acid, during the incubation of several days following the mutation thus an extremely effective enrichment step for those mutants that increase citric acid

; s form, offers a selection advantage over weaker producers. This observation was used as a principle for the method according to the invention.
Yeast mutants which excrete more citric acid than the wild type could only be found by testing a large number of colonies isolated after mutation in fermentation experiments.
According to the invention it has now been possible to achieve this extremely

} s time- consuming procedure, known with uncertain chances of success, to reduce to a minimum of work.

According to the invention, the procedure is that after the mutation has been triggered with the usual known mutagenic Agents such as nitrite, t-methyl-3-nitro-l-nitroso-guanidine, UV or X-rays transfer the treated cell suspension into a glucose mineral medium is inoculated and one in the course of the subsequent, multi-day incubation at 28 ° C with increasing

4s concentrations of aconitase inhibitors or citric acid antimetabolites or their precursors, such as halogenated mono-, di- and tricarboxylic acids, their salts or amides, in particular monofluoroacetate, up to a final concentration of 5-10 " ² mol / l.

M> During this time there is a strong accumulation of mutants with a high level of citric acid excretion, which are then plated out on complete medium plates can be isolated by the usual method. This enrichment technique offers a

> 5 considerable savings in time and effort and increases the chance of discovering suitable mutants considerably, since the only alternative is the investigation a large number of inoculated colonies in fermentation experiments would come into question, the

(χ) desired mutants only an extremely small number Represent the percentage of the total cells present after the mutation has been triggered.

Using this technique, it was possible to isolate mutants of the yeast Candida oleophila, which differed against the

'' s original strain was characterized by a greatly increased excretion of citric acid in carbohydrate media.
When these mutants were used in n-paraffin fermentation media, it was surprisingly found that not here

only the yield of citric acid was increased, but also the percentage of iso-citric acid formed at the same time of approx. 40% of the total acid Original strain had decreased to 0.5 to 5% depending on the mutant strain.

example 1 a) Obtaining suitable mutants

Candida oleophila ATCC No. 20 177, a common yeast which produces citric acid, was removed from an agar slant in 25 ml of glucose-mineral medium (1% glucose. 0.05% KH ₂ PO ₄ , 0.02% MgSO ₄ . 7 H ₂ O, 0.025% MnSO ₄ · 4 H ₂ O, 0.2% NH ₄ Cl, 0.05% yeast extract, 0.1% NaHCO ₃ ) and shaken for 48 hours at 28 ° C. A second glucose-mineral culture was inoculated with this preculture in such a way that the initial cell concentration was approx. After incubation for 15 hours with shaking at 28 ° C., the cells were in the logarithmic growth phase and had reached a concentration of about 8 μl of OVmI. 5 ml of the growing culture were mixed with 0.05 ml of 1-nitrose-3-nitro-1-methyl guanidine solution (50 mg / ml) and shaken for a further 2 hours. During this time, 99.9% of the yeast cells were killed. The reaction was stopped by centrifuging off and washing the cells with 0.9% NaCl solution. The cells were then suspended in complete medium (5% glucose, 0.5% peptone, 0.3% yeast extract, 03% malt extract) and shaken at 28.degree

During the next 5-day incubation, the cells were transferred daily to fresh complete medium with increasing amounts of mono-fluoroacetate to a final concentration of 5 χ 10 ^-2 mol / l. The cell suspensions were then spatially diluted onto complete medium plates with a spatula. The surviving cells grew into colonies within 2 days and were then seeded with different concentrations of fluoroacetate by transferring stamps to glucose-mineral-agar plates. After incubation for 2 days, the plates were sprayed with a cell suspension of the original strain (approx. 10 ⁷ cells / ml in glucose-mineral solution) and incubated for a further 2 days.

On the plates with a fluoroacetate concentration of 10 ^-2 mol / l of the wild-type cells did not come to the growth; However, previously grown, strongly citric acid-secreting mutants could be recognized in a surrounding courtyard of satellite colonies of the original strain, and they could be inoculated and isolated at the appropriate point from the complete medium plate.

Following the procedure described in Example 177, the method 20 mutant AC 7, DSM 343 (German collec-. ₅ Göttingen _s development of microorganisms) was prepared from the yeast strain Candida oleophila ATCC obtained.

Except for the yeast Candida oleophila can according to the invention all the yeasts which are able to produce citric acid, f converted according to Example \ in yeast mutants with high Citronensäurebildungsvermögen, are _0th Thus, according to Example 1, for. B. Mutants of the original strains of Candida lipolytica ATCC 8661, 8662 and 9773 can be obtained.

The following examples are intended to show the technical progress achieved when using the yeast mutants according to the invention in comparison to the original strain in the production of citric acid 5 will.

Example 2

The mutant AC 7 of the original strain Candida oleophila ATCC No. 20 177, which was isolated by the method described in Example 1, was inoculated from an agar slant with yeast-malt extract-peptone-glucose agar in 50 ml of a liquid medium of the same composition and inoculated for 24 hours at 28 The well-grown culture suspension was then transferred to 500 ml of the following medium: 0.05% KH ₂ PO ₄ , 0.02% MgSO ₄ · 7 H ₂ O, 0.025% MnSO ₄ · 4 H ₂ O, 0, 25% NH ₄ CI, 0.05% com steep powder, 2% (vol.) N-dodecane, 1% CaCO ₃ . After 48 hours of cultivation, a second preculture with a total volume of 51 was inoculated with this preculture and cultivated in a fermenter vessel with stirring and supply of air for a further 24 hours.

0.050 / o KH ₂ PO ₄

0.02% MgSO ₄ · _{7H 2} O 0.025% MnSO ₄ · _{4H 2} O

0.25% NH ₄ CI

0.05% powdered corn steep water

8.0% CaCO ₃

10.0% (w / v) n-dodecane

The fermentation was carried out in a 50 l fermenter vessel at 28 ° C. with a stirrer speed of 600 rpm and an air flow rate of 0.7 l / l / min. After 24 hours of cultivation, a maximum cell count of approx. 8 10 ⁸ ZmI was reached.

After 4 days of fermentation, the citric acid concentration in the fermentation medium was 114 / g / l. 4.5 g / l iso-citric acid could be detected.

After adding hydrochloric acid to the medium and filtering off the yeast cells, the citric acid was able to pass through Precipitation with calcium hydroxide and subsequent filtration as tricalcium citrate isolated from the medium and obtained in crystalline form from the concentrated filtrate after reaction with sulfuric acid.

Example 3

Candida oleophila ATCC 20 177 was taken from an agar slant containing yeast-malt extract-peptone-glucose agar in a liquid medium of the same Inoculated composition and shaken at 28 ° C for 24 hours. This cell suspension was used for inoculation a preculture with 2% n-dodecane, the composition of which was described in Example 1, used and after aeration for 48 hours, 10% is transferred into 100 ml of the fermentation medium listed in Example 1. The fermentation took place in a 1 l round bottom flask Shake at 28 ° C.

After 4 days of fermentation, the following concentration ratios were obtained: 72.5 g / l citric acid, 50.5 g / l iso-citric acid.

Claims (1)

Claim: Process for the production of yeast with high citric acid production capacity, in which citric acid-producing yeasts are treated with mutation-inducing agents, the treated yeasts are inoculated into a glucose-mineral medium, then incubated for several days at 28 ° C and the enriched yeast mutants from the nutrient medium with high citric acid-forming ability after plating on solid medium plates by the usual method isolated, characterized in that increasing in the course of several days of incubation at 28 ° C concentrations of Citronensäureantimetaboliten or their precursors to a final concentration of 5 χ 10 ~ ² moles of added / l.